Top > Search of International Patents > MOLECULAR ASSEMBLY CONTAINING MODIFIED RNA, AND RNA DELIVERY SYSTEM USING SAME

MOLECULAR ASSEMBLY CONTAINING MODIFIED RNA, AND RNA DELIVERY SYSTEM USING SAME

Foreign code F160008828
File No. (S2015-0127-N0)
Posted date Aug 10, 2016
Country WIPO
International application number 2015JP081352
International publication number WO 2016072500
Date of international filing Nov 6, 2015
Date of international publication May 12, 2016
Priority data
  • P2014-227611 (Nov 8, 2014) JP
Title MOLECULAR ASSEMBLY CONTAINING MODIFIED RNA, AND RNA DELIVERY SYSTEM USING SAME
Abstract The present invention principally addresses the problem of providing a novel molecular assembly containing functional RNA and capable of introducing functional RNA such as siRNA and shRNA into a cell by in vivo administration, and also delivering the functional RNA to a diseased site. The present invention provides, for example, a molecular assembly characterized by containing the following as essential components thereof: an amphiphilic block polymer A having a hydrophilic block containing a sarcosine chain and a hydrophobic block containing a lactic acid chain; RNA modified by a compound having a long-chain hydrophobic group such as a polylactic acid chain or a polylactic acid-polysarcosine copolymer chain; a plasma-membrane-binding compound (CPP, etc.); and a photosensitizer which may bond to one or more of these components. The present invention is useful as an RNA delivery system (DDS) into tumor cytoplasm and as a cancer preventive or therapeutic agent.
Outline of related art and contending technology BACKGROUND ART
Which is a kind of water soluble drug, a nucleic acid such as RNA or DNA to the backbone in the medicament is a nucleic acid having, in the gene of any particular sequence bonding to the base acts, high specificity, and it has been difficult to treat cancer, such as the treatment of genetic diseases are expected to be applied to. Is a conventional low-molecular compound, a particular candidate compounds that bind to the target needs to be selected for, excessive effort to search for a compound, the probability is only about one-half 30000 marketed in 1. On the other hand, nucleic acid pharmaceutical agents, disease-related gene itself directly target disease etiology and for the expression of the protein can be suppressed, as compared to the low molecular weight compounds are believed to be more versatile. A representative example of the pharmaceuticals siRNA (small interfering RNA) is, base pair 21-23 and double-stranded RNA, called interference RNA, a phenomenon in which mRNA and inhibit the expression, physiological 2006 year Nobel Nobel also is assumed that the prize is medical. However, all over the world pharmaceuticals which resulted in the approval so far is extremely small. The large factor is, in vivo instability of the nucleic acid molecule of the natural immune response are believed to cause induction of side effects. That is, a nucleic acid molecule efficiently in vivo target tissue, carries the cells, its effect is for achieving a safe and effective, and suitable pharmaceutical delivery technique, a so-called DDS (drug delivery system) is required (non-patent document 1, reference 2).
Functional RNA such as siRNA into a cell as a technique for the delivery of a DDS, a cationic liposome containing RNA has been known a technique (for example, see Patent Document 1). Cationic liposomes, the lipid is positively charged and for its configuration, and are negatively charged by electrostatic interaction RNA readily complexes can be formed, and the complex is a fusion with the cell membrane, penetrating into the cells considered RNA. In addition, in the same manner as cationic liposome, the cationic polymer by electrostatic interaction to couple RNA, to be introduced into a cell RNA DDS technique is known (for example, see Patent Document 2) are. However, the electrostatic and cationic polymers such as RNA and a combination method has the problem, the conditions of a living body (such as sulfated polysaccharides) or inhibited by a cationic molecule, attached to the cationic polymer itself within the cells, generally have the ability to enter the highly cytotoxic can be cited.
In recent years, hydrophobic polylactic acid chain and a hydrophilic polysarcosine chain amphiphilic block polymer and a polymer nano-micelles are composed, as being excellent DDS effect have been proposed. Non-patent document 3 and patent document 3 is, the hydrophobic block of polylactic acid chain, polysarcosine hydrophilic block chain of the straight-chain amphiphilic block copolymers, to self-assemble in an aqueous solution, particle size of 30 nm or more of polylactic acid - sarcosine to form a polymeric micelle system disclosed.
Non-patent document 4 is, the hydrophobic block is poly L- lactic acid (PLLA) chain, polysarcosine hydrophilic block chain of the straight-chain amphiphilic block polymer having a poly-lactic acid - sarcosine-based polymer to the micelles, the stereochemistry of the 3 type of indocyanine green (ICG) different labeled polylactic acid (ICG-PLLA, ICG-PDLA, and ICG-PDLLA) and each encompassing, 3 - sarcosine of polylactic acid to prepare a polymeric micelle system disclosed, within the living organism on the stereochemistry of the polylactic acid labeled ICG behavior is disclosed. Non-patent document 4 polymeric micelles used in the particle diameter of the 35 nm or more.
3 And patent document 3 and non-patent document 4 disclosed using a straight-chain amphiphilic block polymer micelles sarcosine - polylactic acid-based polymers, and also has a high blood residing, has already been developed up to the polymer micelles and integrated to the liver as compared with the significantly reduced amount have been reported. In addition, these polylactic acid-based polymer micelles - sarcosine, stays in the blood, of the particle diameter of several tens to several hundred nm - nanoparticles significantly increased vascular permeability tumor (cancer) tissue and inflammation and cause it to accumulate at a site (Enhanced Permeation and Retention effect (EPR effect) ) by utilizing, targeted to a tumor or inflammation or molecular imaging can be used as a carrier for the delivery of medication in the nanoparticles.
Patent Document 4 is, the amphiphilic block polymer, a plurality of the hydrophilic block is comprised of a chain sarcosine have a branch structure by molecular design, self-assembly of the polymer molecules formed assembly is disclosed. Patent Document 4 is a functional material is polylactic acid chain due to the function of a molecular assembly, and a straight-chain in the molecular assembly can be mixed with the amphiphilic polymer is a technique of controlling the particle diameters by the disclosed. Is Patent Document 5, sarcosine and branched chain hydrophilic block, hydrophobic block and the polylactic acid chain branched-type amphiphilic block polymer A, and functional sites and polylactic acid chain including a functional substance F the molecular assembly is disclosed. In the molecular assembly according to the patent document 3, the hydrophobic block of the amphiphilic block polymer A constituting the polylactic acid chains composed of lactic acid unit L -, the functional material contained in the polylactic acid chain F D- whether a lactic acid unit, or, the hydrophobicity of the amphiphilic block polymer A block D- polylactic acid chains composed of lactic acid unit, wherein the functional substance contained in the polylactic acid chain F L- configured from the lactic acid unit. And the like are described in the patent document 3-5, for intracellular delivery of RNA not referred. That is, the molecular assembly described in Patent Document 3-5 is, effective intracellular delivery of RNA is determined to be unknown.
On the other hand, the functional RNA introduced into cells as a technique, a cell-membrane permeable peptide (CPP) and RNA-binding protein (RBP) protein and the carrier, the carrier protein N terminal or C-terminal side connected to the, the near infrared region with light having a wavelength function (PST) photosensitizer having the structure of the carrier molecule is and has been proposed (see Patent Document 6). To the carrier molecule bound RBP RNA in contact with the complex to cells, the near-infrared light is irradiated, the RNA complex is in the cytoplasm and diffused, the functions of the expressed RNA. This technique is, within a cytoplasm RNA is forced into, but function to be produced, the RNA complex may be administered in a living body, the body is readily degraded by the degrading enzyme (RNase) and it may be necessary.
Scope of claims (In Japanese)[請求項1]
次の1~4に記載の成分を必須として含むことを特徴とする、分子集合体。
1)サルコシン鎖を含む親水性ブロックと乳酸鎖を含む疎水性ブロックとを有する両親媒性ブロックポリマーA
2)長鎖疎水性基を有する化合物により修飾されているRNA
3)細胞膜結合性化合物
4)上記1~3の一以上の成分に結合していてもよい光増感剤。
[請求項2]
両親媒性ブロックポリマーAが、20~300個のサルコシン単位を含む親水性ブロックと、10~100個の乳酸単位を含む疎水性ブロックとを有するものである、請求項1に記載の分子集合体。
[請求項3]
長鎖疎水性基を有する化合物が、ポリ乳酸、ポリ乳酸・ポリサルコシンコポリマー、又はジメトキシトリチルオキシ-ヘキシルジチオヘキサンである、請求項1又は2に記載の分子集合体。
[請求項4]
ポリ乳酸又はポリ乳酸・ポリサルコシンコポリマーのポリ乳酸が、10~60個の乳酸単位からなり、ポリサルコシンが、0~100個のサルコシン単位からなるものである、請求項3に記載の分子集合体。
[請求項5]
RNAが、遺伝子発現抑制効果を有するものである、請求項1~4のいずれか一項に記載の分子集合体。
[請求項6]
遺伝子発現抑制効果を有するRNAが、siRNA、shRNA、miRNA、アンチセンスRNA、アプタマーRNA、リボザイムである、請求項5に記載の分子集合体。
[請求項7]
siRNAが、ATP-binding cassette transporter G2(ABCG2)遺伝子をノックダウンし、その発現を抑制するsiRNA、若しくはフェロケラターゼ遺伝子をノックダウンし、その活性を阻害するsiRNAであるか、又はその両者併用物である、請求項6に記載の分子集合体。
[請求項8]
ABCG2遺伝子をノックダウンし、その発現を抑制するsiRNAが、次のセンス鎖及びアンチセンス鎖からなる二本鎖核酸である、請求項7に記載の分子集合体。
センス鎖(配列番号10):
5’-CGAUAUGGAUUUACGGCUUdTdT-3’
アンチセンス鎖(配列番号11):
5’-AAGCCGUAAAUCCAUAUCGdTdG-3’
[請求項9]
フェロケラターゼ遺伝子をノックダウンし、その活性を阻害するsiRNAが、次のセンス鎖及びアンチセンス鎖からなる二本鎖核酸である、請求項7に記載の分子集合体。
センス鎖(配列番号12):
5’-GCAUUUACCAGUGACCAUAdTdT-3’
アンチセンス鎖(配列番号13):
5’-UAUGGUCACUGGUAAAUGCdTdA-3’
[請求項10]
細胞膜結合性化合物が、細胞膜透過性ペプチドである、請求項1~9のいずれか一項に記載の分子集合体。
[請求項11]
細胞膜透過性ペプチドが、
配列番号1:GALFLGFLGAAGSTMGAWSQPKKKRKV
配列番号2:YGRKKRRQRRRG
配列番号3:RRRRNRTRRNRRRVR
配列番号4:YGRRARRRRRRR
配列番号5:KETWWETWWTE
配列番号14:RKKRRRESRKKRRRESC
配列番号15:YARAAARQARAC
配列番号16:KETWWETWWTEWSQPKKKRKVC、又は
配列番号17:LIRLWSHLIHIWFQNRRLKWKKKC
である、請求項10に記載の分子集合体。
[請求項12]
光増感剤が、450nm~1300nmの波長を有する光で機能するものである、請求項1~11のいずれか一項に記載の分子集合体。
[請求項13]
450nm~1300nmの波長を有する光で機能する光増感剤が、フルオレセイン系色素、インドシアニン色素等のシアニン系色素、ローダミン系色素、ポルフィリン系色素、Alexa Fluor(登録商標)546、Alexa Fluor(登録商標)633、Alexa Fluor(登録商標)750、DY750、DY751、DY780、エオシン、ローズベンガル、IRDye(登録商標)800CW、カルボキシフルオレセイン(FAM)である、請求項12に記載の分子集合体。
[請求項14]
粒子径が10~100nmである、請求項1~13のいずれか一項に記載の分子集合体。
[請求項15]
請求項1~14のいずれか一項に記載の分子集合体を用いることを特徴とする、腫瘍細胞質内へのRNA送達システム。
[請求項16]
請求項1~14のいずれか一項に記載の分子集合体を含むことを特徴とする、癌の予防剤又は治療剤。
[請求項17]
請求項16に記載の予防剤又は治療剤、及びそれに含まれる分子集合体中の光増感剤を励起させるための励起光を照射する手段を備えた装置を含む、癌の予防又は治療システム。
[請求項18]
請求項16に記載の予防剤又は治療剤を生体内に投与すること、及び投与した予防剤又は治療剤に含まれる分子集合体中の光増感剤を励起させるための励起光を照射することを含む、癌の予防方法又は治療方法。
[請求項19]
ポリ乳酸又はポリ乳酸・ポリサルコシンコポリマーを有する高分子により修飾されているRNA。
[請求項20]
RNAが、遺伝子発現抑制効果を有するものである、請求項19に記載のRNA。
[請求項21]
遺伝子発現抑制効果を有するRNAが、siRNA、shRNA、miRNA、アンチセンスRNA、アプタマーRNA、リボザイムである、請求項20に記載のRNA。
[請求項22]
siRNAが、ATP-binding cassette transporter G2(ABCG2)遺伝子をノックダウンし、その発現を抑制するsiRNA、若しくはフェロケラターゼ遺伝子をノックダウンし、その活性を阻害するsiRNAであるか、又はその両者併用物である、請求項21に記載のRNA。
[請求項23]
ABCG2遺伝子をノックダウンし、その発現を抑制するsiRNAが、次のセンス鎖及びアンチセンス鎖からなる二本鎖核酸である、請求項22に記載のRNA。
センス鎖(配列番号10):
5’-CGAUAUGGAUUUACGGCUUdTdT-3’
アンチセンス鎖(配列番号11):
5’-AAGCCGUAAAUCCAUAUCGdTdG-3’
[請求項24]
フェロケラターゼ遺伝子をノックダウンし、その活性を阻害するsiRNAが、次のセンス鎖及びアンチセンス鎖からなる二本鎖核酸である、請求項22に記載のRNA。
センス鎖(配列番号12):
5’-GCAUUUACCAGUGACCAUAdTdT-3’
アンチセンス鎖(配列番号13):
5’-UAUGGUCACUGGUAAAUGCdTdA-3’
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NATIONAL UNIVERSITY CORPORATION OKAYAMA UNIVERSITY
  • SHIMADZU CORPORATION
  • Inventor
  • OHTSUKI Takashi
  • MATSUURA Eiji
  • KOBUCHI Hirotsugu
  • AKAHOSHI Akiya
  • OZEKI Eiichi
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KN KP KR KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG

PAGE TOP

close
close
close
close
close
close